molecular biology of the parathyroid · alexander kel department of research and development...

MOLECULAR BIOLOGY INTELUGENCE UNIT

Molecular Biology of the Parathyroid

Tally Naveh-Many, Ph.D. Minerva Center for Calcium and Bone Metabolism

Nephrology Services Hadassah Hebrew University Medical Center

Jerusalem, Israel

L A N D E S B I O S C I E N C E / EUREKAH.COM K L U W E R ACADEMIC / PLENUM PUBLISHERS

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MOLECULAR BIOLOGY OF THE PARATHYROID

Molecular Biology Intelligence Unit

Landes Bioscience / Eurekah.com Kluwer Academic / Plenum Publishers

Copyright ©2005 Eurekah.com and Kluwer Academic / Plenum Publishers

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Molecular Biobgy of the Parathyroid^ edited by Tally Naveh-Many, Landes / Kluwer dual imprint / Landes series: Molecular Biology Intelligence Unit

ISBN: 0-306-47847-1

While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data

Molecular biology of the parathyroid / [edited by] Tally Naveh-Many. p. ; cm. ~ (Molecular biology intelligence unit)

Includes bibliographical references and index. ISBN 0-306-47847-1 1. Parathyroid glands—Molecular aspects. 2. Parathyroid hormone. I. Naveh-Many, Tally. II. Series:

Molecular biology intelligence unit (Unnumbered) [DNLM: 1. Parathyroid Glands—physiology. 2. Molecular Biology. 3. Parathyroid Glands—physiopathol-

ogy. 4. Parathyroid Hormone-physiology. WK 300 M718 2005] QP188.P3M654 2005 6 l 2 . 4 ' 4 - d c 2 2

2004023419

To Dani, Assaf, Yoav and Amir

CONTENTS

Preface . x i i i

1. Development of Parathyroid Glands 1 Thomas Gunther and Gerard Karsenty

Physiology of the Parathyroid Glands 1 Development of Parathyroid Glands in Vertebrates 1 Genetic Control of Parathyroid Gland Development 3

2. Parathyroid Hormone, from Gene to Protein 8 Osnat Belly Justin Silver and Tally Naveh-Many

The Prepro PTH Peptide 8 Homology of the Mature PTH 9 The PTH mRNA 10 Cloning of the PTH cDNAs 11 Homology of the cDNA Sequences 12 Structure of the PTH mRNA 18 The PTH Gene 21 The 5' Flanking Region 24 The 3' Flanking Region 24 Chromosomal Location of the Human PTH Gene 25

3. Toward an Understanding of Human Parathyroid Hormone Structure and Function 29 Lei Jin, Armen H. Tashjian, Jr., and Faming Zhang

PTH and Its Receptor Family 29 PTH Structural Determination 30 Structural Based Design of PTH Analogs 37

4. The Calcium Sensing Receptor 44 Shozo Yano and Edward M. Brown

Biochemical Characteristics of the CaR 45 Disorders Presenting with Abnormalities in Calcium

Metabolism and in the CaR 47 Signaling Pathways of the CaR 49 Drugs Acting on the CaR 50

5. Regulation of Parathyroid Hormone mRNA Stability by Calcium and Phosphate 57 Rachel Kilav, ]ustin Silver and Tally Naveh-Many

Regulation of the Parathyroid Gland by Calcium and Phosphate 57

Protein Binding and PTH mRNA Stability 58 Identification of the PTH mRNA 3'-UTR Binding

Proteins and Their Function 61 Identification of the Minimal cis Acting Protein Binding

Element in the PTH mRNA 3'-UTR 62 The Structure of the PTH rw Acting Element GA

6. In Silico Analysis of Regulatory Sequences in the Human Parathyroid Hormone Gene 68 Alexander KeU Maurice Scheer and Hubert Mayer

Global Homology of PTH Gene between Human and Mouse 71

Computer Assisted Search for Potential Cis-Regulatory Elements in PTH Gene 75

Phylogenetic Footprint: Identification of TF Binding Sites by Comparison of Regulatory Regions of PTH Gene of Different Organisms 78

Discussion 80

7. Regulation of Parathyroid Hormone Gene Expression by 1,25-Dihydroxyvitamin D 84 Tally Naveh'Many and Justin Silver

Transcriptional Regulation of the PTH Gene byl,25(OH)2D3 84

Calreticulin and the Action of l,25(OH)2D3 on die PTH Gene 89

PTH Degradation 90 Secondary Hyperparathyroidism and Parathyroid

Cell Proliferation 90

8. Vitamin D Analogs for the Treatment of Secondary Hyperparathyroism in Chronic Renal Failure 95 Alex J. Brown

Pathogenesis of Secondary Hyperparathyroidism in Chronic Renal Failure 95

Treatment of Secondary Hyperparathyroidism 96 Mechanisms for the Selectivity of Vitamin D Analogs 104 Future Perspectives 109

9. Parathyroid Gland Hyperplasia in Renal Failure 113 Adriana S. Dusso, Mario Cozzolino andEduardo SUtopolsky

Parathyroid Tissue Growth in Normal Conditions and in Renal Failure 114

Dietary Phosphate Regulation of Parathyroid Cell Growth in Uremia 116

Vitamin D Regulation of Uremia- and High Phosphate-Induced Parathyroid Cell Growth 120

Calcium Regulation of Uremia-Induced Parathyroid Growth 123

10. Molecular Mechanisms in Parathyroid Tumorigenesis 128 Eitan Friedman

Oncogenes Involved in Parathyroid Tumor Development 129 Tumor Suppressor Genes Involved in Parathyroid

Tumorigenesis 130 Other Molecular Pathways Involved in Parathyroid

Tumorigenesis 132

11. Molecular Genetic Abnormalities in Sporadic Hyperparathyroidism 140 Trisha M. ShaUuck Sanjay M. Mallya and Andrew Arnold

Implications of the Monoclonality of Parathyroid Tumors 141 Molecular Genetics of Parathyroid Adenomas 142 Molecular Genetics of Parathyroid Carcinoma 151 Molecular Genetics of Secondary and Tertiary

Hyperparathyroidism 152

12. Genetic Causes of Hypoparathyroidism 159 Rachel L Gafni and Michael A. Levine

Disorders of Parathyroid Gland Formation 159 Disorders of Parathyroid Hormone Synthesis or Secretion 167 Parathyroid Gland Destruction 170 Resistance to Parathyroid Hormone 171

13. Skeletal and Reproductive Abnormalities in P/A-Null Mice 179 Dengshun Miao, Bin He, Beate Lanske, Xiu-YingBai,

Xin-Kang Tong, Geoffrey N. Hendy, David Goltzman and Andrew C. Karaplis Results 180 Discussion 188 Materials and Methods 193

Index 197

EDITOR

Tally Naveh-Many Minerva Center for Calcium and Bone Metabolism

Nephrology Services Hadassah Hebrew University Medical Center

Jerusalem, Israel Chapters 2, 5, 7

CONTRIBUTORS Andrew Arnold Center for Molecular Medicine University of Connecticut Health Center Farmington, Connecticut, U.S.A. Chapter 11

Xiu-Ying Bai Division of Endocrinology Department of Medicine and Lady Davis

Institute for Medical Research Sir Mortimer B. Davis-Jewish General

Hospital McGill University Montreal, Canada Chapter 13

Osnat Bell Minerva Center for Calcium

and Bone Metabolism Nephrology Services Hadassah Hebrew University

Medical Center Jerusalem, Israel Chapter 2

Alex J. Brown Renal Division Washington University School

of Medicine St. Louis, Missouri, U.S.A. Chapter 8

Edward M. Brown Endocrine-Hypertension Unit Brigham and Women's Hospital Boston, Massachusetts, U.S.A. Chapter 4

Mario Cozzolino Renal Division Washington University School


Adriana S. Dusso Renal Division Washington University School


Eitan Friedman Institute of Genetics Sheba Medical Center Tel Hashomer, Israel Chapter 10

Rachel I. Gafni Division of Pediatric Endocrinology University of Maryland Medical Systems Baltimore, Maryland, U.S.A. Chapter 12

David Goltzman Calcium Research Laboratory

and Department of Medicine McGill University Health Centre

and Royal Victoria Hospital McGill University Montreal, Canada Chapter 13

Thomas Gunther Department of Obstetrics

and Gynecology Freiburg University Medical Center Freiburg, Germany Chapter 1

Bin He Division of Endocrinology Department of Medicine and Lady Davis



Geoffrey N. Hendy Calcium Research Laboratory



Lei Jin Suntory Pharmaceutical Research

Laboratories LLC Cambridge, Massachusetts, U.S.A. Chapter 3

Andrew C. Karaplis Division of Endocrinology Department of Medicine and Lady Davis



Gerard Karsenty Department of Molecular

and Human Genetics Baylor College of Medicine Houston, Texas, U.S.A. Chapter 1

Alexander Kel Department of Research

and Development BIOBASE GmbH Wolfenbiittel, Germany Chapter 6

Rachel Kilav Minerva Center for Calcium


Medical Center Jerusalem, Israel Chapter 5

Beate Lanske Department of Oral and Developmental

Biology Forsyth Institute and Harvard School

of Dental Medicine Boston, Massachusetts, U.S.A. Chapter 13

Michael A. Levine Department of Pediatric Endocrinology The Children's Hospital

at The Cleveland Clinic Cleveland Clinic Lerner College

of Medicine of Case Western Reserve University

Cleveland, Ohio, U.S.A. Chapter 12

Dengshun Miao Calcium Research Laboratory



Sanjay M. Mallya Center for Molecular Medicine University of Connecticut School

of Medicine Farmington, Connecticut, U.S.A. Chapter 11

Hubert Mayer Department of Gene Regulation Gesellschaft fiir Biotechnologische

Forschung Braunschweig, Germany Chapter 6

Maurice Scheer Department of Research

and Development BIOBASEGmbH Wolfenbuttel, Germany Chapter 6

Trisha M. Shattuck Center for Molecular Medicine University of Connecticut School

of Medicine Farmington, Connecticut, U.S.A. Chapter 11

Justin Silver Minerva Center for Calcium


Medical Center Jerusalem, Israel Chapters 2, 5, 7

Eduardo Slatopolsky Renal Division Washington University School


Armen H. Tashjian, Jr. Department of Cancer Cell Biology Harvard School of Public Health

and Department of Biological Chemistry and Molecular Pharmacology

Harvard Medical School Boston, Massachusetts, U.S.A. Chapter 3

Xin-BCang Tong Division of Endocrinology Department of Medicine and Lady Davis



Shozo Yano Department of Nephrology Ichinomiya Municipal Hospital Ichinomiya, Aichi, Japan Chapter 4

Faming Zhang Lilly Research Laboratories Eli Lilly & Company Indianapolis, Indiana, U.S.A. Chapter 3

PREFACE

M aintaining extracellular calcium concentrations within a narrow range is critical for the survival of most vertebrates. PTH, together with vitamin D, responds to hypocalcemia to increase extracellu

lar calcium levels, by acting on bone, kidney and intestine. The recent introduction of PTH as a major therapeutic agent in osteoporosis has directed renewed interest in this important hormone and in the physiology of the parathyroid gland. The parathyroid is unique in that low serum calcium stimulates PTH secretion. As hypocalcemia persists, there is also an increase in PTH synthesis. Chronic hypocalcemia leads to hypertrophy and hyperplasia of the parathyroid gland together with increased production of the hormone. Phosphate is also a key modulator of PTH secretion, gene expression and parathyroid cell proliferation.

Understanding the biology of the parathyroid as well as the mechanisms of associated diseases has taken great strides in recent years. This book summarizes the molecular mechanisms involved in the function of the parathyroid gland. The first chapter reviews the development of the parathyroid gland and the genes involved in this process as identified using genetically manipulated mice. Then the biosynthetic pathway of PTH from gene expression to its intracellular processing and the sequences in the gene controlling its transcription as well as those regulating mRNA processing, stability and translation are described. Studies on the structure of PTH with correlations to its function are presented and provide a starting point for understanding the recognition of the PTH ligand by its receptor the PTH/PTHrP or PTHl receptor. The calcium sensing receptor regulates PTH secretion, gene expression and parathyroid cell proliferation. A chapter on the calcium receptor focuses on the signalling pathways that it activates and the associated disorders that involve the calcium receptor gene and lead to excess or decreased PTH secretion. Calcium and phosphate regulate PTH gene expression post-transcriptionally. The mechanisms of this regulation and the cis and trans acting factors that are involved in determining PTH mRNA stability are described. Vitamin D s active metabolite, l,25(OH)2-vitamin D3, regulates PTH gene transcription. The regulatory sequences in the human PTH gene and the studies on the regulation of PTH gene transcription by 1,2 5 (OH)2-vitamin D3 as well as the subsequent use of vitamin D analogs for the treatment of secondary hyperparathyroidism are all reviewed. Patients with chronic renal failure develop excessive activity of the parathyroid gland that causes severe bone disease. The known factors involved in its pathogenesis are 1,25(OH)2-vitamin D3, a low serum calcium and a high serum phosphate. Insights into the mechanisms implicated in secondary hyperparathyroidism of renal failure are now being revealed and are discussed. Additional chapters are devoted to the pathophysiology of

abnormalities of the parathyroid. The genetic alterations involved in parathyroid tumorigenesis are summarized. In addition, the genetic causes of sporadic hyperparathyroidism and hypoparathyroidism are reviewed. The genetic mutations leading to diseases of hyper- or hypoactivity of the parathyroid have elucidated a host of interacting transcription factors that have a central role in normal physiology. Finally, the last chapter focuses on the characteristics of PTH-nuU mice and the skeletal and reproductive abnormalities that they present.

Together the chapters of this book offer a state of the art description of the major aspects of the molecular biology of the parathyroid gland, PTH production and secretion. The book is designed for students and teachers as well as scientists and investigators who wish to acquire an overview of the changing nature of the PTH field. I would like to express my deep appreciation to all the authors who have contributed to this book for their comprehensive and stimulating chapters and for making the book what it is. I am especially grateful to Justin Silver for his help and support that have made this book possible. I also thank Landes Bioscience for giving me the opportunity to edit this book.

Tally Naveh'Many, Ph.D.

CHAPTER 1

Development of Parathyroid Glands

Thomas Gtinther and Gerard Karsenty

Summary

The parathyroid glands (PG) are the main source for circulating parathyroid hormone (PTH), a hormone that is essential for the regulation of calcium and phosphate metabolism. The PGs develop during embryogenesis from the pharyngeal pouches

with contributions from endodermal and neural crest cells. A few genes have been attributed to the formation, migration and differentiation of the PG anlage. In studies mostly done in genetically manipulated mice it could be demonstrated that Rae28, Hoxa3, Paxly Pax9 and Gcm2 are essential for proper PG formation. Recently, candidate genes involved in the DiGeorge syndrome have been identified as well.

Physiology of the Parathyroid Glands The parathyroids are small glands located in the cervical region in close proximity to the

thyroids. The main function of the PGs is the secretion of PTH. It is on top of a complex hormonal cascade regulating serum calcium concentration (Fig. 1). The latter is remarkably constant in diverse organisms under various physiological conditions. This tight regtdation is important since calcium is essential for many functions such as muscle contraction, neuronal excitability, blood coagulation, mineralization of bone and others. A reduction of the serum calcium concentration to less than 50% will lead to tetany and subsequendy to death. The importance of a strict regulation of the serum calcium is also reflected by the rapid secretion of PTH within seconds, new synthesis of the hormone within minutes and new transcription within hours following a decrease in serum calcium concentration which is detected through the calcium sensing receptor expressed in the PGs. The overall role of PTH is to increase calcium concentration. It fulfils this function through three different means. First it prevents calcium elimination in the urine, second it favors the hydroxylation in one of the 25 hydroxycholecalciferol and as a results it favors indirectly intestinal calcium absorption. Lastly PTH favors through still poorly understood mechanisms bone resorption and as a result increases the extracellular calcium concentration (Fig. 1).

Development of Parathyroid Glands in Vertebrates The PGs derive from the pharyngeal pouches which are transient structures during em

bryonic development. They are evolutionary homologous to gill slits in fish. The foregut endo-derm and cells originating from the neural crest of rhombomere 6 and 7 contribute to the anlage of the PGs. The neural crest originates at the apposition of neuroectoderm and ectoderm during the formation of the neural tube. Therefore neural crest cells have to migrate

Molecular Biology of the Parathyroid, edited by Tally Naveh-Many. ©2005 Eurekah.com and Kluwer Academic / Plenum Publishers.


Parathyroid Hormone PTH

reabsorption Hydroxy lation of 25(OH) vitamin D

Figure 1. Regulation of calcium homeostasis. Parathyroid hormone is on top of a hormonal cascade regulating serum calcium concentration. PTH secretion leads to an increase of serum calcium through renal reabsorption and intestinal absorption, the latter is caused by the induaion of the synthesis of the active form of vitamin D in the kidney. Bone is the main reservoir for calcium containing more than 99% of the body content. Calcium is released through bone resorption. The main source for circulating PTH are the parathyroid glands (PG) while P /̂?-expressing cells in the thymus can funaion as a backup in mice.

towards the foregut endoderm first before they can add to the anlage of the PGs. Neural crest of rhombomere 6 migrates towards the third branchial arch while the fourth branchial arch is primarily invaded by neural crest cells from rhombomere 7 (Fig. 2).

Mice only have one pair of PGs deriving from the third pharyngeal pouch homologous to the inferior PGs in men while the superior ones derive from the fourth pharyngeal pouch. The anlage of the PGs in mice first becomes visible between embryonic day 11 ( E l l ) and El 1.5 histologically in a very limited area in the dorsal region of the cranial wall of the third endoder-mal pouch while the caudal portion of the very same pouch develops into the thymus which is involved in the maturation of the immune system (Fig. 2). Both domains are demarkated by the complementary expression of Gcm2 and Foxnl (the latter mutated in nude mice, lacking a functional thymus), respectively already two days before the anlagen are morphologically visible."^ In contrast to thymus development, induction of the ectoderm is not necessary for the formation of the PGs.^ In mammals both structures start to migrate shortly thereafter towards the caudal end before at around E l 4 they seperate. While the thymus moves on further in the direction of the heart the PGs become incorporated to the thyroid gland between El 4 and El 5.


Figure 2. Specification of the parathyroid gland anlage. The parathyroid glands develop from the third pahryngeal pouch (in humans from P3 and P4). Neural crest cells evaginating from rhombomere six and seven (R6, R7) of the hindbrain and pharyngeal endoderm contribute the primordium of PGs and thymus. Both anlagen are demarcated by the expression of Gcm2 and Foxnly respectively, already two days before the anlagen become histological visible. The identity of the neural crest is determined by genes of the Hox cluster. The anterior expression borders oi Hoxa/b3 and Hoxh4 are depicted.

Pth is expressed already in the anlage of the PGs at E l 1.5 and contributes to fetal serum calcium regulation to some extent although placental transport involving parathyroid hormone related protein (PTHrP) is more important. The parathyroid gland is not the only source of P T H . T h e protein is also synthesized by a few cells in the hypothalamus and in the thymus. It has been shovv^n in mice that the thymic /V/?-expressing cells actually contribute to the circulating hormone keeping the level of serum calcium even in the absence of PGs at a concentration compatible with life.

Genetic Control of Parathyroid Gland Development Three different steps can be used to separate the formation of the PGs mechanistically.

They include (I) formation of the PGs, (II) migration towards their final destination and (III) the difFerentiation towards P T H producing cells (Fig. 3). Mouse mutants that highlight the role of the few genes known to be involved in these different processes have been generated in the last decade.


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M i 8 r a t i 0 n

I) i f f e r e n t i a t i 0 1)

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MiOXUJ

i Pax9

Hoxa3 ^

Paxl I

\ J Gem 2

Pth

fc Z^,-, , . , • • >

W UCfHZ

Rae28

Shh

I Thxl

Figure 3. Schematic representation of parathyroid gland development. Parathyroid gland development can be mechanistically seperated into formation of the anlage, caudal migration towards their final location within the thyroid glands and differentiation into PTH-secreting cells. The genetic interactions between factors involved in induction, maintenance, specification and fiinction are shown.

Both, neural crest cells and the pharyngeal endoderm contribute to the anlage of the PGs. Neural crest cells possibly already maintain information about their localization along the anterior-posterior axis before they start to migrate ventrally. They derive this information from a group of evolutionary conserved transcription factors containing a homebox, the Hox genes, organized in four paralogous genomic clusters (Hoxa, b, c and d). Hox genes are expressed in the neural crest prior to, during and after migration into the pharyngeal arches and endodermal epithelia express Hox genes as well.

I. Rae28 is the mouse homologue of the Drosophila polyhomeotic gene which is required for the proper expression of hometic genes along the anterior-posterior axis. Similar, absence of Rae28 causes an anterior shift of anterior expression boundaries of several genes of the Hox cluster including Hoxa3, Hoxb3 and Hoxb4. Mice deficient for Rae28 are characterized by malformations of tissues partly derived from neural crest like altered localization of PGs as well as PG and thymic hypoplasia and cardiac anomalies.^ How the altered hox expression pattern influences PG formation still needs to be evaluated. The first reported malformation of PGs caused by a deletion through homologous recombination in mouse embryonic stem cells were represented by //ttv/23-deficient animals. Among other defects knockout mice are devoid of PGs and thymus and exhibit thyroid hypoplasia.^ This coincides very well with Hoxa3 expression in the third and fourth pharyngeal arches and in the pharyngeal endoderm. The Hoxa3 signal does neither effect the number of neural crest cells nor their migration pattern. Mutant cells rather lost their capacity to induce differentiation of surrounding tissues.̂ ®


Absence of the paired box containing transcription factor Pax9 in targeted mice also displays absence of PGs and thymus. Pax9 is expressed in the pharyngeal endoderm. The epithelial buds separating from the third pharyngeal pouch did not form in the mutant mice. This phenotype could be traced back to delayed development of the third pouch already at El 1.5 and coincides with the expression oi Pax9 in the pharyngeal endoderm.^

II. PGs develop normally in mice deficient for the paralogous Hoxb3 and Hoxd3. However further removal of a single Hoxa3 allele leads to the inability of the normally formed anlge of the PGs to migrate to their position next to the thyroid gland. ̂ ^ Therefore, development and migration of the PGs are separable events which is consistent with the fact that in other vertebrates like fish and birds PGs do not migrate fi-om location of their origination.

III. Glial cell missing2 (Gcm2) is the homoloug of the Drosophila GCM transcription factor. Unlike its glia cell fate determining function in fruit flies implies, mouse Gcm2 exclusively characterizes parathyroid cells and starts to be expressed around ElO in the pharyngeal endoderm. ̂ ^ The pattern rapidly becomes restricted to the cranial portion of the third pharyngeal pouch.-^ Mice deficient for Gcm2 revealed that PTHh never expressed in the PG anlage although parathyroid like cells characterized by Pax9 expression are still present at El4.5.'^ This clearly points out that Gcm2 is essential for the specification of precursors to become /V/'-expressing cells rather than for the induction of the precursors itself (Fig. 3). Interestingly, Pth-positive cells still could be detected in the thymus of mutant mice indicating that at least 2 pathways for the specification of/V/^-expressing cells exist (Fig. 1). Gcml expressed in the thymus is the most likely candidate to compensate for Gcm2 function. It will be compelling to determine if a ,backup mechanism* for the parathyroid gland also exists in man. In this direction it is very interesting to note that the first human homozygous mutation for GCM2\\2iS been identified in hypoparathyroidic patients.̂ -^

It has been discovered just recendy that newborn P<ayf7-deficient mice exhibit severely reduced PGs.^^ The reduction in size could be traced back to the beginning of PG development at El 1.5. The hypoplasia of the anlage was even more severe in Hoxa3-\-/-Paxl-/-embryous and PGs were absent at late gestational stages. ̂ ^ Interestingly, Gcm2 expression although properly initiated at El0.5 was reduced at El 1.5 in P43;xr7-deficient embryos while the reduction was even more severe in the compound mutant. //(9x^3-deficient embryos exhibit no Gcm2 signal at all.̂ ^ Therefore, Hoxa3 is necessary for Gcm2 induction while both Hoxa3 and Paxl are substantial for the proper maintenance of Gcm2 expression. Paxl expression in the PG primordium on the other hand is reduced in //(9Ar^3-deficient mice.̂ '^^ This would place Hoxa3 genetically upstream oiPaxl and both upstream of Gr;w2 which in turn is required for PTT/expression in PGs (Fig. 3).

A long time known conglomerate of congenital malformations in humans including dysplasia or absence of the PGs and thymus as well as malformations of the heart outflow is the DiGeorge syndrome. The organs affected derive in part from neural crest so that mutations in one or several genes influencing these cells have been suspected to be the cause for the disease. It could be shown that most patients are hemizygous for a megabase deletion on chromosome 22ql 1. Recently, two groups came up with a good candidate eene for several of the features in DiGeorge syndrome including PG defects simultaneously. ' Both laboratories generated hemizygous megabase deletions comprising more than a dozen genes on the synthenic mouse chromosome 16 that reflected the human malformations including PG abnormalities. TBXl was among them and it could be shown that the gene is expressed in the pharvngeal endoderm and mesoderm-derived core but not in neural crest-derived mesenchyme.^ ' Tbxl expression in the pharyngeal arches is possibly induced through the morphogen Sonic hedgehog.^ Mice heterozygous for a Tbxl deletion by homologous recombination reflected the pharyngeal arch artery malformations while homozygous-deficient mice exhibited PG hypoplasia. ' ' ^


DiGeorge syndrome patients resemble hemizygous deletions. This suggests that other genes of this region may contribute to the PG phenotype. Indeed, Guris and colleagues^^ could demonstrate that mice homozygous for a targeted null mutation for Cr^o/dysplay cardiovascular, PG and thymus defects. The migration and early proliferation of neural crest cells was not altered pointing out that Crkol influences the function of neural crest during later stages. CRKL (ho-molog human gene name) also maps within the common deletion region for the DiGeorg syndrome.

Deletions on chromosome lOp also cause DiGeorge like malformations. The locus includs a subregion that encodes for the hypoparathyroidism, sensorineural deafness, renal anomaly (HDR) syndrome. Van Esch and her colleagues^^ could demonstrate that two heterozygous patients exhibit loss of function mutations in GATA3. The transcription factor is indeed expressed in the affected organs during human and mouse embryonic development. Surprisingly though, heterozygous knockout mice have been reported to be normal while homoyzgous mice die around El2.^^

The understanding of the contribution from several gene products to the development of PGs from these critical regions still awaits further analysis.

Concluding Remark Clinical studies indicate that multiple mutations can account for the malfunction of se

rum calcium regulation through PTH in humans. These include the synthesis of PTH, sensing of the calcium content in the blood stream as well as the development PGs and proper specification of PTH translating cells. It is astonishing how rather litde is known so far on the molecular level in comparison to the formation of other organs. Surely, the genome sequencing projects for mice and man and the use of microarrays to compare different cDNA pools will shed new light on this issue in the near future.

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3. Manley N R and Capecchi MR The role of Hoxa-3 in mouse thymus and thyroid development.

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4. Giinther T, Chen ZF, Kim J et al. Genetic ablation of parathyroid glands reveals another source of

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velo-cardio-facial/DiGeorge syndrome. Cell 2001; 104: 619-629.

15. Lindsay EA, Vitelli F, Su H et al. Tbx l haploinsufficieny in the DiGeorge syndrome region causes

aortic arch defects in mice. Nature 2001; 410: 97-101.

16. Garg V, Yamagishi C, H u T et al. T b x l , a DiGeorge syndrome candidate gene, is regulated by

sonic hedgehog during pharyngeal arch development. Dev Biol 2001; 235:62-73.

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gene, T b x l . Nat Genet 2001; 27:286-291.

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phenocopy neurocristopathies of DiGeorge syndrome. Nat Genet 200; 27: 293-298.

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CHAPTER 2

Parathyroid Hormone, from Gene to Protein

Osnat Bell, Justin Silver and Tally Naveh-Many

Abstract

The biosynthetic pathway of parathyroid hormone (PTH) has been studied from gene expression to PTH intracellular processing.' The processing of PTH has been described and involves the synthesis of an initial translational product, preProPTH, and two

proteolytic cleavages that in turn produce Pro PTH and PTH. The genes and cDNAs from ten different species have been cloned, sequenced and characterized. This chapter will summarize the molecular biology of PTH, from the gene to the mRNA, the initial translational product, preProPTH and the processed mature secreted form of PTH. It will describe the sequences of the PTH gene and mRNA in different species and the specific elements in the PTH mRNA that determine mRNA processing, stability and translation.

The Prepro PTH Peptide The primary form of PTH, which is stored and secreted, contains 84 amino acids. PTH

is initially synthesized as a precursor, preProPTH. Two proteolytic cleavages produce the ProPTH and the secreted form of PTH. The proPTH sequence contains six extra amino acids at the N-terminus.^' Conversion of ProPTH to PTH occurrs about 15 to 20 min after biosynthesis at about the time ProPTH reached the Golgi apparatus.^

The Structure of the Pre-Peptide Evidence that the translational product of PTH mRNA was larger than ProPTH was

initially obtained by translation of a crude preparation of bovine parathyroid RNA in the wheat germ cell-free system. The primary translational product migrated slower than ProPTH when analyzed by electrophoresis on either acidic-urea or sodium dodecyl sidfate-containing acrylamide gels. At that time, a similar phenomenon had been observed only for myeloma light chains.'^ In further studies, preProPTH was shown to be synthesized in cell-free systems of reticulocyte lysates.^ Translation of human parathyroid RNA also produced an analogous preProPTH.^

The observation that the carboxyl terminal peptides of bovine PTH and preProPTH were identical indicated that the extra amino acids in preProPTH were at the amino terminus. This was confirmed by incorporating selected radioactive amino acids into preProPTH and determining the location of the radioactivity by automated Edman degradation.'^ By analyzing overlap of these radioactive amino acids with those in ProPTH, the length of the bovine pre-peptide was shown to be 25 amino acids. The entire sequence of the bovine pre-peptide was determined eventually by this microsequencing technique'' and was later confirmed by

Molecular Biology of the Parathyroid^ edited by Tally Naveh-Many. ©2005 Eurekah.com and Kluwer Academic / Plenum Publishers.

Parathyroid Hormone, from Gene to Protein

structural studies of both the bovine PTH cDNA and gene. ' The sequence of human pre-peptide was also partially determined by this microsequencing technique.^ The complete amino acid sequence was derived from the human PTH cDNA sequence ̂ ^ and later confirmed by the determination of the structure of the human gene.^ The amino acid sequence of the rat pre-peptide was derived from the sequence of the rat PTH gene^^ and partially by analysis of cloned rat PTH cDNA.^^

The amino acid sequences of the pre-pep tides show that the human and bovine pre-pep tides are 80% homologous while the rat sequence is GA% homologous to the bovine and human. ̂ This is somewhat lower than the homology of 89 and 77% in the Pro and PTH regions for bovine/human and rat/bovine-human, respectively (Fig. 1). The fact that the pre-peptide is less conserved than the rest of the molecule is consistent with pre-peptides or signal peptides of many eukaryotic proteins.^^ General structural features of the signal peptides are a central hydrophobic core and, in many cases, charged amino acids at the N-terminal and C-terminal ends of the central core. These features are largely retained in the pre-peptides of the three preProPTH molecules. Only conservative changes are present within the central core of uncharged amino acids from amino acids 10 to 21.^

Conversion ofPrePro to ProPTH The removal of the pre-peptide to produce ProPTH is mediated by an enzyme associated

with microsomes. In reticidocyte and wheat germ systems that contain litde or no microsomal membranes, the primary transcriptional product of PTH mRNA is preProPTH. ' Addition of microsomal membranes from dog pancreas or chicken oviduct results in the synthesis of ProPTH.^'20

The first evidence that pre or signal peptides fiinction by binding to a limited number sites in the microsomal membrane was obtained by studies on a synthetic prePro-peptide of bovine preProPTH. The identification of the signal recognition particle as a signal peptide receptor, later on, confirmed this mechanism for most secreted and membrane proteins.

The pre peptide of preProPTH is rapidly degraded after its proteolytic cleavage from preProPTH. In studies of PTH biosynthesis in intact cells, no labeled pre-peptide could be detected. The proteolytic removal of the pre-peptide probably occurs before completion of the ProPTH nascent chain, since preProPTH is difficult to detect in intact cells.

Homology of the Mature PTH The mature PTH has been determined or predicted by the cDNAs in several species. The

sequence of PTH of mouse, rat, man, non-human primates, horse, dog, cat, cow, pig, and chicken is shown in Figure 1. The resulting phylogenetic tree obtained from alignment of the protein sequences is shown in Figure 3A.

A comparison of the amino acid sequences of PTH from several species revealed high conservation of the protein amongst all species apart from gallus (Fig. 1). In addition, three relatively conserved regions could be observed. The first two regions comprise the biologically active region of PTH and would be expected to be conserved. The addition or loss of a single amino acid at the amino terminus gready reduces biological activity, and the region is involved in binding of PTH to the receptor. In addition there is a region of conservation at the C-terminal region that is itself of interest, particularly since this region may have a separate biological effect at least on osteoclasts. Analyses of the silent changes that occur between the nucleotide sequences suggest that the conservation in the C-terminal region may be related to pre-translational events. Analysis by Perler et al described replacement changes that result in changes in amino acids and silent changes that do not alter the encoded amino acid.

10 Molecular Biology of the Parathyroid

murine rat human macaca Equine canine feline bovine porcine gallus



1 MMSANTVAKV MMSASTMAKV MIPAKDMAKV MIPAKDMAKV

MMSAKDMVKV MMSAKDMVKV MMSAKDMVKV MMSAKDTVKV MTSTKNLAKA

51 RMQWLRRKLQ RMQWLRKKLQ RVEWLRKKLQ RVEWLRKKLQ RVEWLRKKLQ RVEWLRKKLQ RVEWLRRKLQ RVEWLRKKLQ RVEWLRKKLQ RQDWLQMKLQ

101 GNPKS GNSKS SHEKS SHEKS SHQXS SYQKS NHQKS SHQKS SHQKS

EHLRAAVQKK

MIIMLAVCLL MILMLAVCLL MIVMLAICFL MIVMLAICFL

MIVMFAICFL MVVMFAICFL MIVMLAICFL MVVMLAICFL IVILYAICFF

DMHNFVSLGV DVHNFVSLGV DVHNFVALGA DVHNFIALGA DVHNFIALGA DVHNFVALGA DVHNFVALGA DVHNFVALGA DVHNFVALGA DVHS

LGEGDKADVD LGEGDKADVD LGEADKADVN LGEADKADVD LGEADKADVD LGEADKADVD LGEADKADVD LGEADKADVD LGEADKAAVD SIDLDKAYMN

pre

1 TQTDGKPVRK TQADGKPVKK TKSDGKSVKK TKSDGKSVKK

K AKSDGKPVKK AKSDGKPVKK ARSDGKSVKK ARSDGKPIKK TNSDGRPMMK

QMAARDGSHQ QMAAREGSYQ PLAPRDAGSQ PLAPRDAGSQ PIFHRDGGSQ PIAHRDCSSQ PIAHRDGGSQ SIAYRDGSSQ SIVHRDGGSQ ..ALEDARTQ

128 VLVKSKSQ VLVKAKSQ VLTKAKSQ VLTKAKSQ VLSKTKSQ VLTKAKSQ VLIKAKSQ VLIKAKPQ VLIKAKPQ VLFKTKP-

pro

1 RAVSEIQLMH RAVSEIQLMH RSVSEIQLMH RSVSEIQLMH RSVSEIQLMH RSVSEIQFMH RSVSEIQFMH RAVSEIQFMH RSVSEIQLMH RSVSEMQLMH

KPTKKEENVL RPTKKEENVL RPRKKEDNVL RPRKKEDNIL RPRKKEDNVL RPLKKEDNVL RPRKKEDNVP RPRKKEDNVL RPRKKEDNVL RPRNKEDIVL

50 NLGKHLASME NLGKHLASVE NLGKHLNSME NLGKHLNSME NLGKHLNSVE NLGKHLSSME NLGKHLSSVE NLGKHLSSME NLGKHLSSLE NLGEHRHTVE

100 VD VD VE VE IE VE AE VE VE GEIRNRRLLP

Figure 1. Alignment of the amino acid sequences of PTH from the 10 different species. Alignments were obtained using the default setting of PileUp program (Accelrys Inc. Madison WI). Comparison of the amino acid sequences of PTH for mouse (mus), rat, human, non human primates (macaca), horse (equine), dog (canine), cat (feline), cow (bovine), pig (porcine) and chicken (gallus). Gaps indicated by dashes were introduced to maximize the homology to the gallus sequence. The N terminal sequence of the equus PTH is not available. The arrows indicate the protolytic cleavage sites required for the conversion of preProPTH to ProPTH and PTH.

The PTH mRNA Bovine preProPTH mRNA was initially more extensively characterized than the mRNAs

from the other species. Preparations of bovine parathyroid RNA were obtained that contained about 50% PTH mRNA as estimated by gel electrophoresis and RNA excess hybridization to radioactive cDNA."^ The size of the mRNA was estimated to be about 750 nucleotides by sucrose gradient centrifiigation. About two thirds of the translatably active mRNA was retained by oligo(dT) cellulose, and the sizes of the poly(A) extension was broadly distributed around an average size of 60 adenylate residues, though this may be an under estimation of the actual size. While not direcdy determined, PTH mRNA probably contains a 7-methylguanosine cap since the translation of PTH mRNA was inhibited by 7-methylguanosine-5 -phosphate. The human and bovine

molecular biology of the parathyroid · alexander kel department of research and development...

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